Field of the Invention
This invention relates to an apparatus for separating and analyzing DNA, RNA and proteins, and in particular relates to a capillary array electrophoresis apparatus for determining the base sequences of DNA and RNA, and measuring the polymorphism of the base sequences of DNA based on the diversity of individual base sequences.
The analysis of DNA and RNA is becoming increasingly important in genetic analysis and medicine including genetic diagnosis and biology. In recent years, in particular, high-speed, high throughput DNA analyzers are being developed in connection with genome analysis plans.
In DNA analysis, a fluorephore-labeled sample is separated according to molecular weight by gel electrophoresis, and the fluorescence of the fluorephore label is detected. In gel electrophoresis, a flat plate gel formed by polymerizing acrylamide between two glass plates separated by an interval of approximately 0.3 mm is widely used (Biotechnology 6, 816 (1988)). A sample injected at the upper end of the flat plate gel is caused to move by electrophoresis towards the lower end while undergoing molecular separation due to a voltage applied between the two ends of the flat plate gel. A position which is electrophoretically at a certain distance is then irradiated by a laser which irradiates the whole electrophoresis path from the side face of the flat plate gel, and the separated components of the fluorephore-labeled sample passing through the laser irradiation part are excited. The fluorescence from the fluorephore-labeled sample is continuously and periodically measured at a fixed time interval. The results are analyzed to determine a DNA base sequence.
Recently, instead of a flat plate gel, a capillary gel, i.e., a polymerized gel in a fused quartz glass capillary tube, has come to be used. Capillary gel electrophoresis is attracting attention as a larger electric field can be applied than in slab gel electrophoresis, thereby permitting high-speed analysis (Analytical Chemistry 62, 900 (1990)). Normally, an on-column fluorescence detection measurement is performed wherein one capillary tube is used, and the vicinity of the lower end of the capillary is irradiated by a laser. The entire outer surface of the capillary has a polyimide coating, and the coating is removed at the position where fluorescence is to be detected so that the glass is exposed to form a window (U.S. Pat. No. 5,312,535). When this window position is irradiated by the laser, separated components of a fluorephore-labeled sample subject to electrophoresis in the capillary tube are excited when they pass through the beam, the fluorescence from the fluorephore-labeled sample is measured, and is then analyzed to determine the DNA base sequence.
However, in the aforesaid on-line column measuring apparatus, there were disadvantages such as considerable scattering of the laser beam on the outer surface of the capillary, moreover the capillary can be used only once and throughput could not be increased. Recently, there have been several reports of high throughput capillary array electrophoresis devices wherein plural capillaries are disposed in an array, and a large number of samples are simultaneously analyzed at high speed.
The first report is a capillary array scanning method (Nature, 359, 167 (1992)). Plural capillaries are irradiated in sequence one at a time, and on-column fluorescence detection is performed. The fluorescence detection positions of the plural capillaries are disposed horizontally in a plane, a laser beam converged by a lens from a perpendicular direction to the plane is irradiated to one capillary in the array, and the fluorescence is detected by a light collecting lens on the side of the laser beam light source. The part of the capillary array in the plane is moved back and forth in a perpendicular direction to the axis of the capillaries, and laser irradiation and fluorescence detection are sequentially performed for each capillary. The laser irradiation system and fluorescence receiving optical system are fixed. A construction is adopted with a common focal point where the position at which the laser beam is most converged in the capillaries and the position of the light source incident on the fluorescence measuring instrument coincide, and each capillary is measured independently.
The second report is a multiple sheath-flow method (Nature, 361, 565-566 (1993), Japanese Patent Laid-Open Hei 06-138037 (Koho)). The sample elution end of a capillary array disposed in a plane is vertically immersed in a buffer solution, sample components separated by gel electrophoresis are eluted from the capillaries into the buffer solution, a part where there are no capillaries is irradiated by a laser, and fluorescence is detected. An arrangement is adopted wherein the buffer solution is made to flow gradually in the electrophoresis direction so that separate components eluted from different capillary gels are not mixed together in the buffer solution, or two components separated in one capillary gel are not mixed in the buffer solution. A buffer solution part where there are no capillaries, in the vicinity of the outlet of the capillary array, is irradiated by the laser. Hence, the problem of scattering of the laser beam on the surface of the capillaries is avoided, components eluted from plural capillaries are excited together, and fluorescence detection is performed simultaneously. The fluorescence from all the electrophoresis lanes is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
The third report is a laser beam expansion method (Analytical Chemistry, 66, 1424-1431 (1994)). Fluorescence detection parts of plural capillaries are arranged horizontally in a plane, and a laser beam is irradiated at an angle of 45xc2x0 relative to the axis of the capillaries. The laser beam is enlarged in the perpendicular direction to the capillary axes by a cylindrical lens, and all the capillaries are irradiated simultaneously. Fluorescence from all the capillaries is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
The fourth report is a multiple laser focusing method (Analytical Chemistry, 68, 2699-2704 (1996)). The fluorescence detecting parts of plural capillaries are arranged in a plane, and a laser beam is irradiated from the side of the plane so as to pass through the center of each capillary. As the laser beam is repeatedly converged by the converging action of each capillary and is not dispersed by the capillary array, all the capillaries can be irradiated simultaneously. Fluorescence from all the capillaries is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
In on-column measurement wherein a capillary is directly irradiated by a laser beam and the resulting fluorescence is detected, the reflection of the laser beam from the inner surface and outer surface of the capillary which enters the fluorescence detecting system gives rise to a high level of background light. If a sufficiently converged laser beam is incident perpendicularly to the axis of the capillary and a fluorescence measurement is performed in a perpendicular direction to the plane formed by the laser beam and capillary axis, the intensity of the laser reflection incident on the fluorescence detecting system is of the order of 10xe2x88x923 for a laser reflection intensity of 1. If fluorescence detection is performed in the same direction as that of laser incidence, the laser reflection intensity incident on the fluorescence detecting system is of the order of 10xe2x88x922. On the other hand, the fraction of laser scattering intensity due to the separating medium packed in the interior of capillary which is incident on the fluorescence detecting system is of the order of 10xe2x88x925. In other words, the laser reflection intensity is two to three orders of magnitude larger than the laser scattering intensity, and it is the major component of the background light in fluorescence measurement.
In capillary array scanning, as laser irradiation and fluorescence measurement are performed sequentially for one capillary at a time, the fluorescence detection time for one capillary is less than in ordinary on-column measurement. In the case of an n capillary array, the fluorescence detection time per capillary is a maximum of 1/n, but in practice, a glass part of the capillary through which sample separating components do not pass is also scanned, so this time is less than 1/n. The time interval between adjacent peaks in the sample electrophoresis pattern is smaller the more rapid the analysis, but if it increases to such an extent that the time required for one scan cannot be ignored compared to this time interval, the resolution of the electrophoresis pattern will decrease.
In a capillary array scanning system, there are a large number of moving parts for scanning, and as they move through large distances, the scanning speed is not very high, in addition to which breakdowns often occur. Moreover, as the signal from each capillary is processed separately, i.e., as the signal obtained in one fluorescence detecting time period corresponds to the signal from one capillary, the scanning speed cannot be much increased. Due to these reasons, current scanning speeds are of the order of 1 Hz. Further, as the laser beam is incident perpendicularly to the capillary axis, and fluorescence detection is performed in the same direction as the incidence direction, a large amount of laser light reflected at the capillary surface enters the fluorescence detection system. As described above, this laser reflection intensity is extremely high, so the fluorescence measurement background light increases and the detection sensitivity declines.
The problem of the multiple sheath-flow method is that, in comparison to an on-column fluorescence measurement, the fluorescence intensity obtained from molecular separation components decreases the higher the molecular weight. This problem is due to the following reason. To ensure that separated components eluted into the buffer solution from the lower end of the capillary gel do not mix due to diffusion, etc., in the buffer solution, the buffer solution must constantly be made to flow at a minimum constant rate in the electrophoresis direction. On the other hand, the electrophoresis rate of sample components which move through the capillary gel as their molecular weights are separated, becomes smaller the larger the molecular weight, i.e., the larger the length of DNA base sequences. The lower the electrophoresis rate of the components in the capillary gel compared to the flowrate of buffer solution, the more the sample components are drawn out in the electrophoresis direction when they are eluted into the buffer solution from the capillary gel. As a result, the fluorescence intensity declines, and detection sensitivity decreases.
In the laser beam expansion method, each capillary is irradiated by a uniform laser intensity, so the expansion width of the laser beam in the perpendicular direction to the capillary axis must be greatly increased compared to the width of the capillary array. This is because the intensity distribution of the laser beam is a Gaussian distribution centered on the beam axis. For example, if the laser expansion width is equal to the capillary array width, the laser irradiation intensity of the capillaries at both ends of the capillary array is of the order of 14% of that of the central capillary. To reduce the variation of irradiating laser intensity between capillaries to 10% or less, the laser expansion width must be increased to at least four times the capillary array width. However, the more the laser expansion width is increased, the more the laser irradiating intensity of each capillary decreases, and the more the fluorescence detection sensitivity decreases. To prevent this, a laser source must be used which has higher output than the laser sources which are usually used. This makes the equipment more bulky and costly.
In the multiple laser focusing method, when the laser beam passes through the capillary array, the laser beam is reflected at the outer surface and inner surface of each capillary, and part of this reflected light enters the fluorescence detection system. As described above, this laser reflection intensity is extremely high, so fluorescence measurement background light increases and detection sensitivity falls.
It is therefore an object of this invention to perform an on-column fluorescence measurement wherein all the scanning is performed by the laser beam, and the fluorescence from all the capillaries is measured in one operation without moving the fluorescence detection system, thereby providing a capillary array electrophoresis apparatus wherein the above problems are resolved.
The capillary array electrophoresis apparatus according to this invention comprises plural capillaries for the electrophoretic separation of a fluorephore-labeled sample, fluorescence detecting parts provided in part of the plural capillaries in the same plane for detecting the fluorescence emitted by a fluorephore label irradiated by a laser beam which scans part of the plural capillaries, and a fluorescence detection system for detecting fluorescence.
In a first construction, scanning is performed by the laser beam to repeatedly irradiate the fluorescence detecting part, the scanning period of the laser beam in fluorescence detecting part is t1, and fluorescence is detected by the fluorescence detection system in a fluorescence detection time t2 (t1xe2x89xa6t2). In this construction, the laser beam is irradiated from a direction wherein the maximum value of the angle between the laser beam and the plane in which the capillaries are disposed, is xcex80xe2x89xa690xc2x0, and the fluorescence detection system comprises an objective lens.
In a second construction, if the laser beam is irradiated from a direction wherein the maximum value of the angle between the laser beam and the plane in which the capillaries are disposed, is xcex80xe2x89xa690xc2x0, scanning is performed by the laser beam to repeatedly irradiate the fluorescence detecting part, the scanning period of a laser beam in fluorescence detecting part is t1, and fluorescence is detected by the fluorescence detection system in a fluorescence detection time t2 (t2xe2x89xa6t1) from a direction wherein the angle made with the plane in which the capillaries are disposed, is xcex83xe2x89xa690xc2x0, the relations t1xe2x89xa6t2 and xcex80 less than xcex83xe2x88x92tanxe2x88x921(D/2d) are satisfied when the diameter of entrance pupil of the fluorescence detection system is D, and the distance between the center position of the entrance pupil and the position at which the capillaries are irradiated by the laser beam is d. In this construction, the fluorescence detection system is provided with an objective lens, the central axis of the objective lens makes an angle of xcex83xe2x89xa690xc2x0 with the plane in which the capillaries are disposed, the diameter of entrance pupil of the objective lens is D, and the focal distance of the objective lens is d=f.
In a third construction, if the laser beam is irradiated from a direction wherein the maximum value of the angle between the laser beam and the plane in which the capillaries are disposed, is xcex80xe2x89xa690xc2x0, scanning is performed by the laser beam to repeatedly irradiate the fluorescence detecting part, the scanning period of the laser beam in fluorescence detecting part is t1, the central axis of the objective lens makes an angle of xcex83xe2x89xa690xc2x0 with the capillary axis, fluorescence is detected by the fluorescence detecting system in a fluorescence detection time t2, and the distance d between the center position of the entrance pupil of the fluorescence detection system and the position at which the capillaries are irradiated by the laser beam is equal to the focal length of the objective lens, the relations t1xe2x89xa6t2 and xcex80 less than 180xc2x0xe2x88x92xcex83xe2x88x92tanxe2x88x921(D/2f) are satisfied where the entrance pupil of the objective lens is D.
In a fourth construction, if the laser beam is irradiated from a direction wherein the maximum value of the angle between the laser beam and the plane in which the capillaries are disposed, is xcex80xe2x89xa690xc2x0, scanning is performed by the laser beam to repeatedly irradiate the fluorescence detecting part, the scanning period of a laser beam in fluorescence detecting part is t1, fluorescence is detected by the fluorescence detecting system in a fluorescence detection time t2 from a direction in which an angle made with the plane in which the capillaries are disposed is xcex83xe2x89xa690xc2x0, the relations t1 greater than t2 and xcex80 greater than xcex83+tanxe2x88x921(D/2d) are satisfied where the diameter of the entrance pupil of the objective lens is D, and the distance between the center position of the entrance pupil and the position at which the capillaries are irradiated by the laser beam is d. In this construction, the fluorescence detection system is provided with an objective lens, the central axis of the objective lens makes an angle of xcex83xe2x89xa690xc2x0 with the plane in which the capillaries are disposed, and when the diameter of the entrance pupil of the objective lens is D, and the focal distance of the objective lens is f, d=f.
In all of the above constructions, the fluorescence detecting part may be a transparent liquid or a transparent solid.
The fluorescence detecting parts of the plural capillaries are disposed in a planar arrangement, the laser beam is focused to irradiate one capillary, and the laser beam is made to scan continuously so as to repeatedly irradiate the fluorescence detection position of each capillary in sequence. The laser beam irradiated to each capillary is reflected by the outer surface and inner surface of the capillary, however these reflected beams are not emitted in all directions but only within a limited range. By disposing the effective aperture (entrance pupil) of the fluorescence detection system which measures the fluorescence emitted by a fluorephore label of a sample moving along the capillaries in one operation, so that this aperture lies outside the aforesaid limited range, the reflected beams do not enter the fluorescence detection system. For example, the laser beam may be focused and made to scan continuously from the direction of an angle of 45xc2x0 relative to the capillary axis, and irradiate the fluorescence detecting position of each capillary in sequence. The fluorescence detection system simultaneously measures the fluorescence from all the capillaries in one operation from a perpendicular direction to the plane of the capillary array.
The light of the laser beam reflected from the capillary surface is emitted in a direction making an angle of 45xc2x0 to the capillary axis on the other side of the fluorescence detection system, i.e., laser reflection light is no longer incident on the fluorescence detection system. Further, the time required for the laser beam to perform one scan of the fluorescence detection parts of plural capillaries is arranged to be less than the signal (measuring) acquisition time (fluorescence detection time) in continuous fluorescence measurement. In other words, all the capillaries receive at least one laser irradiation and emit fluorescence in one fluorescence detection (exposure) time period. The scanning array electrophoresis apparatus which implements this laser scanning system offers the following advantages compared to the prior art apparatus.
In capillary array scanning systems, it was physically difficult to increase the scanning rate above 1 Hz. In the construction of this invention, scanning is performed only by the laser beam, there are very few moving parts required for scanning, and moving distances such as that of a galvanomirror, for example, are very small, therefore high-speed scanning is possible. Fluorescence detection is performed simultaneously for all capillaries, so the signal processing rate is sufficiently fast and the resolution of the electrophoresis analysis does not decrease. Also, in the construction of this invention, laser beam reflection light does not enter the fluorescence detection system, so the fluorescence measurement background light level is very low and a high sensitivity fluorescence measurement can be made.
In the construction of this invention, the laser beam is directly irradiated to the capillaries and on-column fluorescence measurement is performed, so decrease of signal intensity with long base sequences, which was a problem in the multiple sheath-flow method, does not occur.
In the laser beam expansion method, the intensity distribution of the laser beam is a Gaussian distribution, so the laser irradiation intensity was different between capillaries, but in the construction of this invention, laser beam scanning is performed at a constant rate, so all capillaries are irradiated by the laser at a uniform intensity. Further, the laser beam scanning width and the width of the capillary array may be made to coincide so that there is no wastage of laser output, and fluorescence measurement may be performed with a laser source of normal output.
In the multiple laser focusing method, part of the laser beam light reflected at the capillary surface entered the fluorescence detection system and caused an increase in background light. However, according to this invention, as described above, a construction is adopted wherein reflected light from the laser beam does not enter the fluorescence detection system, so background light is reduced and a high sensitivity fluorescence measurement can be performed.
According to this invention, the scanning period of the laser beam is arranged to be much shorter than the detection (exposure) period of the fluorescence measurement and scanning is performed with the laser beam to irradiate plural capillaries, so all the capillaries can effectively be simultaneously irradiated, and fluorescence from all the capillaries can be measured in one operation. As the optical system is such that the reflected beam and transmitted beam from the laser irradiated to the capillaries do not enter the entrance pupil of the fluorescence detection system, the background light intensity of the fluorescence measurement is greatly reduced, a high sensitivity fluorescence measurement can be made, and high-speed, high throughput analysis is achieved.
The typical features of this invention will now be summarized referring to FIG. 1. The electrophoresis apparatus according to this invention comprises plural capillaries 1 which perform electrophoretic separation of a fluorephore-labeled sample, fluorescence detecting parts provided in part of these plural capillaries disposed in the same plane which detect the fluorescence emitted by the fluorephore label irradiated by a laser beam 3 which scans part of the plural capillaries, and a fluorescence detection system 10 which detects fluorescence. Scanning is performed by the laser beam so as to repeatedly irradiate the fluorescence detecting parts, the scanning period of a fluorescence detecting part is t1, and fluorescence is detected by the fluorescence detecting system in a fluorescence detection time t2 (t1xe2x89xa6t2). The laser beam 3 from a laser source 2 is narrowly collimated by a light collecting lens 4, a galvanomirror 5 is rotated in a rotational direction 7 of the galvanomirror around a galvanomirror rotation axis 6, and the fluorescence detecting parts are thereby repeatedly scanned. A laser reflection beam 8 and transmitted beam do not enter the fluorescence detection system. According to the above construction, the effect of background light is reduced, and a high sensitivity, large dynamic range fluorescence measurement can be performed.